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Miltenyi Biotec distribution:

As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is
committed to providing our customers around the world with the highest quality products.
In addition to direct selling in more than 20 countries in North America, Europe and
Asia/Pacific, Miltenyi Biotec also provides support for our customers through an
extensive distributor network covering dozens of additional countries.

As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is
committed to providing our customers around the world with the highest quality products.
In addition to direct selling in more than 20 countries in North America, Europe and
Asia/Pacific, Miltenyi Biotec also provides support for our customers through an
extensive distributor network covering dozens of additional countries.

NK cells

1 Introduction

Natural killer (NK) cells are part of the innate immune system and mediate responses against viruses, parasites, bacteria, and tumor cells. Additionally, NK cells contribute to the adaptive immune response by linking innate and the adaptive immunity through their receptor FcγRIIIA (CD16).

2 NK cells from peripheral blood

Two distinct subsets of human NK cells have been identified based on CD56 expression: CD56dim and CD56bright NK cells (PMID: 3086432). These NK cell subsets lack CD3 expression and are phenotypically and functionally distinct. We also include natural killer T (NKT) cells in this chapter as they also express the CD56 marker on their surface – however they do express CD3. Additional NK cell subsets can be identified according to their tissue distribution. These have a different repertoire and effector functions compared to peripheral blood NK cells.

The majority of human peripheral blood NK cells are CD56dim (90%) and express high levels of CD16; a minority of the cells (10%) are CD56bright and CD16dim/neg.

CD56bright NK cells are known for their capacity to produce and secrete cytokines, such as granulocyte–macrophage colony-stimulating factor (GM-CSF), IFN-γ, interleukin (IL)-10, IL-13, and TNF-β. Resting CD56bright and CD56dim NK cell subsets show differences in their NK cell receptor repertoires (PMID: 9469418). CD56bright NK cells express the high/intermediate affinity IL-2 receptor that confers the capacity to expand in vitro and in vivo in response to low doses of IL-2 (PMID: 7678599, 1692080). CD56bright NK cells do not express CD16 and express low levels of CD69, killer-cell immunoglobulin-like receptors (KIRs), and intracellular perforin (PMID: 15536127). Resting cells have low cytotoxicity, but after activation with IL-2 or IL-12, CD56bright cells exhibit similar or enhanced cytotoxicity against targets compared to CD56dim cells.

CD56dim NK cells are considered terminally differentiated and mature NK cells that are enriched in bone marrow, blood, and spleen (PMID: 12480696, 15536127, 16606675). CD56dim NK cells are cytolytic and efficient effectors of natural and antibody-dependent target cell lysis. In contrast to CD56bright NK cells, resting CD56dim NK cells express only the intermediate-affinity IL-2 receptor and proliferate weakly in response to high doses of IL-2 in vitro (PMID: 1370410). An important feature of CD56dim NK cells is the expression of KIRs and CD16 but not CCR7 and L-selectin. CD56dim cells also express perforin and granzymes, and are more cytotoxic against NK cell–sensitive targets (K562 and COLO205 cell lines) than CD56bright NK cells (PMID: 2530273).

2.3 Sample preparation of peripheral blood

Dedicated solutions from Miltenyi Biotec allow the direct isolation of NK cells from whole blood, but they can also be isolated from PBMCs generated by density gradient centrifugation or using the MACSprep™ PBMC Isolation Kit, human. Other starting materials such as whole blood, buffy coat or cone, leukocyte reduction system chamber (LRSC), and leukapheresis products can also be used to isolate NK cells. For more detailed information, see the MACS Handbook chapter Human blood.

MACS Handbook:

2.4 Magnetic cell separation of peripheral blood NK cells

Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of CD56+ cells. CD56+ cells can be isolated either straight from whole blood, buffy coat, or LRSC without density gradient centrifugation and erythrocyte lysis, or from PBMCs after density gradient centrifugation.

The StraightFrom® CD56 MicroBead Kits were developed for the rapid selection and isolation of CD56+ cells directly from whole blood, buffy coat, or LRSC. The kits require no sample preparation and are optimized for a specific starting material. Subsequent depletion of CD3+ cells using CD3 MicroBeads, human yields purified NK cells.

StraightFrom® Buffy Coat CD56 MicroBead Kit

Before separation

Enriched CD56+ cells

Fast isolation of CD56+ cells from buffy coat. Separation was performed with the StraightFrom® Buffy Coat CD56 MicroBead Kit, human and the MultiMACS™ Cell24 Separator Plus in combination with the Single-Column Adapter and Whole Blood Columns. Cells were fluorescently stained with CD56-PE, CD3-APC, CD45-VioBlue® and analyzed by flow cytometry on the MACSQuant® Analyzer. Cells were triggered via CD45-VioBlue, cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

MACSxpress® NK Cell Isolation Kit, human

Before separation

After separation

Untouched NK cells from whole blood. The MACSxpress NK Cell Isolation Kit, human, a MACSmix™ Tube Rotator, and a MACSxpress Separator were used to separate NK cells from 30 mL of human EDTA-anticoagulated whole blood. The isolated cells were fluorescently stained with CD45-VioBlue, CD3-FITC, and CD56-PE, and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.

Instead of working directly with whole blood or whole blood products, one can process the samples by density gradient centrifugation to pre-enrich peripheral blood mononuclear cells (PBMCs). PBMCs then serve as starting material for NK and NKT cell isolation by the following options.

Pure population of non-activated NK cells. In a first step, the REAlease CD56 MicroBead Kit was used to enrich CD56+ cells. After removal of the REAlease MicroBeads, the CD3+ cell fraction was depleted from the CD56+ cell population with CD3 MicroBeads, human to yield CD56+CD3– NK cells with a high purity of 98%. Monitoring CD69 and CD25 levels by flow cytometry after isolation confirmed no activation of the NK cells.

Pure population of non-activated NK cells.

In a first step, REAlease CD56 MicroBeads were used to enrich CD56+ cells. After removal of the REAlease MicroBeads, the CD3+ cell fraction was depleted from the CD56+ cell population with CD3 MicroBeads, human to yield CD56+CD3– NK cells with a high purity of 98%. Monitoring CD69 and CD25 levels by flow cytometry after isolation confirmed no activation of the NK cells.

Isolation of untouched human NK cells from PBMCs. Samples were processed with the NK Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with CD56-PE, CD3 FITC, and CD45-VioBlue, and then analyzed by flow cytometry on the MACSQuant Analyzer. Cells were triggered via CD45-VioBlue. Debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Isolation of untouched human NK cells from human PBMCs

Samples were processed with the NK Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with CD56-PE, CD3 FITC, and CD45-VioBlue, and then analyzed by flow cytometry on the MACSQuant Analyzer. Cells were triggered via CD45-VioBlue. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

2.4.3 Isolation of NK cell subsets from PBMCs

At a glance: Kits and reagents for the separation of NK cells from PMCs

The table summarizes dedicated kits for the effective isolation of different NK cell subsets. The new REAlease CD56 MicroBead Kit, human however enables the isolation of virtually any NK cell subset. The kit is designed to isolate CD56+ cells (NK and NKT cells) by positive selection. Magnetic labeling can then be easily removed for subsequent separation steps based on other markers.

2.5 Characterization of peripheral blood NK cells by flow cytometry

NK cell subsets and their differentiation status can be determined by flow cytometry based on their expression of cell surface markers, transcription factors, and their secretion of cytokines. Miltenyi Biotec offers a vast portfolio of conventional and REAfinity™ Recombinant Antibodies for comprehensive analysis.

2.5.2 Analysis of cytokines and transcription factors

MACS Cytokine Secretion Assays allow detection and enrichment of viable cytokine-secreting cells. The kits are available for a wide range of cytokines, including TNF-α, GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, and IL-22, and can be used for further characterization of NK cell subsets.

The MACSPlex Cytokine Kits are used for multiplex analysis of secreted cytokines in serum and cell culture supernatants using a standard flow cytometer. Cytokines that can be analyzed in a single sample include: GM-CSF, IFN-α, IFN-γ, lL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.

Principle of MACSPlex Assays. Samples containing unknown levels of analytes are incubated with MACSPlex (MPx) Capture Beads coated with analyte-specific antibodies. Consequently, analytes bind to their corresponding MPx Capture Bead. A Detection Reagent, composed of a cocktail of APC-conjugated antibodies specific for the analytes, is added. Consequently, sandwich complexes are formed between the MPx Capture Bead, the analyte and the Detection Reagent. These complexes can be analyzed based on the fluorescence characteristics of both the MPx Capture Bead and the Detection Reagent. Standards of known quantities of given analytes are provided with the kit and are used for the quantification of the analytes within the unknown samples.

Principle of MACSPlex Assays.

Samples containing unknown levels of analytes are incubated with MACSPlex (MPx) Capture Beads coated with analyte-specific antibodies. Consequently, analytes bind to their corresponding MPx Capture Bead. A Detection Reagent, composed of a cocktail of APC-conjugated antibodies specific for the analytes, is added. Consequently, sandwich complexes are formed between the MPx Capture Bead, the analyte and the Detection Reagent. These complexes can be analyzed based on the fluorescence characteristics of both the MPx Capture Bead and the Detection Reagent. Standards of known quantities of given analytes are provided with the kit and are used for the quantification of the analytes within the unknown samples.

2.6 Culture of NK cells

NK cells are cultured to assess their cytotoxic functions, to study how to enhance these functions, and to obtain larger numbers of cells for downstream applications, just to name a few applications.

NK MACS Medium, research grade is a cell culture medium developed specifically for NK cells. It has been used in a variety of applications and, in combination with MACS Cytokines, is an ideal starting point for reliable cultivation conditions.

NK cell activation is essential for a variety of downstream application. Miltenyi Biotec offers polyclonal stimulation reagents that have been carefully designed to ensure optimal stimulation conditions.

The NK Cell Activation/Expansion Kit, human employs large cell-sized particles loaded with biotinylated antibodies against CD2 and CD335 (NKp46) to activate and expand primary cells. The large cell-sized particles mimic antigen-presenting cells and, when applied in a specific bead-to-cell ratio, lead to efficient NK cell activation.

NK Cell Activation/Expansion Kit, human

NK cell expansion with the NK Cell Activation/Expansion Kit. Starting with NK cells isolated with the NK Cell Isolation Kit, human (n = 2), cells were stimulated at day 1 using the NK Cell Activation/Expansion Kit, human or left unstimulated by culturing in medium with IL‑2 alone.

3 NK cells from other tissues (lymphoid, non-lymphoid)

Although CD56dim NK cells predominate in blood, CD56bright NK cells are far more abundant in the human body due to their enrichment in lymphoid and non-lymphoid tissues. Their presence, however, varies greatly – from only a small percentage of total lymphocytes in secondary lymphoid organs to up to 50% or more of all lymphocytes in some organs, such as the human uterus (PMID: 27121652).

NK cells are also known to infiltrate tumor tissue to varying degrees, depending on the tumor. For more information about tumor-infiltrating lymphocytes, see Human – Tumor tissue and Cells in tumor tissue.